The invention is a process for producing demineralized osteoinductive bone, and demineralized osteoinductive bone produced thereby. The process achieves demineralization of bone by subjecting bone, including for example ground bone, bone cubes or strips, and essentially intact bone, to a rapid high volume, pulsatile acidification wave process. The process allows bone to be rapidly demineralized to a precise and specific desired residual calcium level, without sacrificing osteoinductivity.
Demineralized freeze-dried bone allograft is widely used in the repair of skeletal defects and periodontal disease. It is known that the implantation of acid demineralized bone in the form of a powder in extraskeletal sites may stimulate new bone formation. Various groups including Syftestad, 1982; Urist et al., 1967; Urist and Strates, 1979; Urist and Strates, 1971; Urist et al., 1983; have suggested that a noncollagenous protein or proteins present in demineralized bone has the ability to induce new bone formation when present within the implanted bone matrix.
Current procedures used to demineralize ground bone involve the use of ethanol to remove lipids and hydrochloric acid to remove the mineral components of bone. These known methods are problematic in that they require prohibitively long periods of time for processing resulting in a very low demineralization rate; require excessive handling of the ground bone being processed; are capable of processing only small amounts of ground bone; and result in a demineralized bone product which exhibits inferior osteoinductivitiy caused by excessive exposure of bone inducing proteins in the bone to harsh acids over extended periods of time.
The invention achieves high demineralization rates by subjecting bone, for example ground cortical bone, to rapid and complete exchanges of acid. Suitable acids include both highly ionized and/or weak acids. The inventors have discovered that the acid neutralization rate of bone mineral apatite is highly dependent upon the bone surface concentration of the acid and the demineralization reaction products. The initial reaction rate of the acid at the surface of the bone particle is very rapid and quickly terminates, due to boundary layer resistance caused by the increasing concentrations of the reaction by-products, if the residual reaction products are not promptly removed. Since the bone is subjected to the demineralizing acid for very brief periods of time, bone-inducing proteins are not adversely affected, thus resulting in a bone product, which achieves maximum potential osteoinductivity. The process also allows the demineralization of an entire single donor""s tissue volume in a single batch.
The invention provides a rapid demineralization process for producing osteoinductive bone. including subjecting bone to two or more pulse and drain exchanges of a predetermined volume of one or more demineralizing acid solutions to produce demineralized bone.
The invention also provides a rapid demineralization process for producing osteoinductive bone, including subjecting bone to two or more pulse and drain exchanges of a predetermined volume of one or more demineralizing acid solutions under conditions effective to produce demineralized bone.
The invention further provides a rapid demineralization process where each of the pulse and drain exchanges, include rapidly pulsing a predetermined volume of one or more demineralizing acid solutions into the container, incubating the bone and the predetermined volume of one or more demineralizing acid solutions in the container for an interval of time, and rapidly draining the predetermined volume of one or more demineralizing acid solutions from the container.
The invention also provides a rapid demineralization process for producing osteoinductive demineralized bone, including placing an amount of bone to be demineralized into a substantially closed container, and cycling the bone in the substantially closed container through two or more pulse and drain cycles to produce osteoinductive demineralized bone, each of the two or more pulse and drain cycles, including rapidly pulsing a predetermined volume of one or more demineralizing acid solutions into the container, incubating the bone and one or more demineralizing acid solutions in the container for an interval of time, and rapidly draining the predetermined volume of one or more demineralizing acid solutions from the container.
The invention provides a rapid demineralization process where the step of incubating includes agitation including for example, stirring, shaking, orbital shaking, and/or sonicating.
The invention provides a rapid demineralization process for demineralizing bone by subjecting bone to two or more rapid fill and drain pulses of one or more of the following: a demineralizing acid, and a cation extractor including for example one or more metal chelators or electrolyte solutions.
The invention provides a rapid demineralization process for demineralizing bone by subjecting bone to two or more changes of one or more demineralizing acids, under conditions sufficient to achieve a demineralization rate of from about 1.5 g demineralized bone per minute to about 30.0 g per minute, preferably form about 5.0 g per minute to about 30.0 g per minute, more preferably from about 8.0 g per minute to about 25.0 g per minute, and most preferably from about 10.0 g demineralized bone per minute to about 22.0 g per minute.
The invention further provides a rapid demineralization process for demineralizing bone where the fill and drain pulses are carried put over intervals of from about 1.0 minutes to about 30 minutes, preferably from about 2.0 minutes to about 25.0 minutes, and more preferably from about 3.0 minutes to about 20.0 minutes, and most preferably about 5.0 minutes.
The invention also provides a rapid demineralization process for demineralizing bone where rapid acid pulse or rapid acid drain is achieved over a time period of from about 0.5 minutes to about 8 minutes, preferably from about 0.5 minutes to about 2.0 minutes to rapidly fill (pulse) the vessel with acid, more preferably about 1.0 minutes to pulse the acid; and preferably about 1.0 to about 10.0 minutes to drain acid, and more preferably from about 2.0 minutes to about 5.0 minutes to drain the acid.
The invention also provides a rapid demineralization process for demineralizing bone by subjecting bone to two or more rapid fill and drain pulses of one or more acid solutions where the bone is demineralized until a specific desired residual calcium level is achieved.